Cleavage of Alzheimer's amyloid precursor protein by alpha-secretase occurs at the surface of neuronal cells

The amyloid precursor protein (APP) is proteolytically processed predominan tly by alpha-secretase to release the ectodomain (sAPP alpha). In this stud y, we have addressed the cellular location of The constitutive alpha-secret ase cleavage of endogenous APP in a neuronal cell line. Incubation of the n euroblastoma cell line IMR32 at 20 degrees C prevented the secretion into t he medium of soluble wild-type APP cleaved by alpha-secretase as revealed b y both immunoelectrophoretic blot analysis with a site-specific antibody an d immunoprecipitation following metabolic labeling of the cells. No sAPP al pha was detected in the cell lysates following incubation of the cells at 2 0 degrees C, indicating that alpha-secretase does not cleave APP in the sec retory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible t o inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S ., et al, (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPP alpha by > 92%, indicating that alpha-secretase is cleaving APP al most exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in th e secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleav ed primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPP alpha.